Antimalarial and Immunomodulatory Activities of Tithonia diversifolia (Asteraceae) Leave Flafonoids-Rich Extract Used in Cameroonian Traditional Medecine

Background Phytochemicals are considered the reliable source for the treatment of infection including malaria. Especially, phenols are known as potentially toxic to the growth and development of pathogens, among which flavonoids are the most extensively studied and play more intensive roles in ethnopharmacology. The immunological effect and role of T. diversifolia flavonoids-rich extract in treatment of malaria have therefore been examined in this study. Methods In vitro test against Plasmodium falciparum and 4-day suppressive and Rane's tests against Plasmodium berghei in mice were used to evaluate the antimalarial activities. TNF-α and INF-γ levels, phagocytic tests, and production of oxygen and nitrogen radical were assessed to appreciate the immunomodulatory activity. One-way analysis of variance followed by post hoc Student's t tests was used for data analysis. Results T. diversifolia flavonoids-rich extract at the concentrations ranging from 0.0004 mg/ml significantly (p < 0.05) inhibited in a concentration-dependent manner the growth of trophozoite up to 100% inhibition with 0.025 mg/ml at 24 and 48 hrs. Moreover, T. diversifolia flavonoids-rich extract reduced the level of parasitemia and improved in a dose-dependent manner the survival time of infected mice significantly (p < 0.05) compared to their control in 4-day suppressive test as well as in Rane's test. Additionally, T. diversifolia flavonoids-rich extract increased the TNF-α and INF-γ levels in rats infected by P. berghei. Furthermore, the flavonoid-rich extract enhanced weight of spleen in the rats, the metabolic and phagocytic activities of the peritoneal cells, and the concentration of nitric oxide and oxygen radicals in methylprednisolone-immunocompromised rats compared to the control (p < 0.05). Conclusion The study has revealed that T. diversifolia flavonoids-rich extract through its antiplasmodial and phagocytic activities is a promising treatment of malaria.


Introduction
Malaria is one of the most devastating mosquito-borne diseases caused by several species of single-celled protozoan parasite known as Plasmodium.It remains an important disease afecting the world population.Consequently, one of the most important pillars of malaria control is the antimalarial therapy [1].In sub-Saharan Africa, the proportion of patients utilizing antimalarial treatment outside the ofcial circuit varies from 12 to 80% [2].Te extensive resistance to chloroquine and later to the antifolate drugs sulfadoxine-pyrimethamine led to the worldwide adoption of artemisinin-(ART-) based combination treatments (ACTs) as the frst-line treatment of uncomplicated P. falciparum malaria in the early 2000s.However, the emergence of ART resistance is of signifcant concern.
Antimalarial drug efcacy is typically monitored by determining the in vivo therapeutic efcacy or in vitro/ex vivo sensitivity.However, in the developing world, people are exposed to the use of herbal decoctions for treatment of malaria.Tey are encouraged by the multiple actions of medicinal plants, directly through their antiplasmodial efect or by stimulating the immune response.Nitric oxide (NO) generated by phagocytes has antimicrobial activity demonstrated to kill P. falciparum in vitro [3].Various antimalarial drugs are suspected to act via the production of reactive oxygen species (ROS) and nitric oxide (NO) production.Terefore, in addition to searching for antimalarial drugs, concerted research studies are also underway to boost the immune response against the parasite.
Over the past decades, the use of medicinal plant has become increasingly important in antimalarial therapy.T. diversifolia is one such plant.Te leaves are traditionally used against malaria by boiling in water, and it is recommended as a candidate for antimalarial screening.In general, phytodrugs are one of the most important pillars of malaria control [4], and plants are of multiple chemical compounds, among which favonoids, the polyphenolic compounds, are found to be highly antimalarial [5].In previous studies, various bioactive compounds from T. diversifolia have been declared for signifcant therapeutic implications and favourable safety index [6].However, from our knowledge, favonoids of that plant were not investigated as antimalarial drugs.In order to identify the phytocompounds of this plant providing the same or better results as traditional healers in malaria treatment, the favonoids-rich extract of leaf was assayed for its antimalarial and immunoregulatory activities.Te results of this study would permit to validate the presence of these favonoids with a potential antimalarial activity in T. diversifolia and the use of the plant as an effective therapeutic means in novel drug discovery.

Experimental Material
2.1.1.Plant Collection and Identifcation.Te fresh leaves of T. diversifolia were collected in the morning hours (9:30 am-10:30 am) from Nkwen, Bamenda III subdivision, in the Northwest Region of Cameroon.Te plant was identifed by botanists in the Department of Biological Sciences, University of Bamenda.Te identifcation was authenticated by the National Herbarium at Yaounde, Cameroon.Te sample was identifed in comparison with the material of Dang Daniel no: 100 of National Herbarium No: 18591 SRF/Cam.Te collected leaves were cleaned, sliced, and air-dried under shade at room temperature.Te dried leaves were coarsely powdered.Ten the powdered plant material was stored in a plastic container at room temperature until extraction.

Extraction of Flavonoids.
Flavonoid-rich extract of leaves from T. diversifolia was prepared as described by Krishnadhas et al. [5].In summary, 10 g of the plant powdered was weighed and discarded into 100 mL of petroleum ether and incubated in an oven (40-60 °C) for 24 hours; this was done to remove the lipids and sugars from the plants.Te solid residue obtained was then treated with ethyl acetate for 24 hours and fltered.Te resulting fltrate was concentrated using fash evaporator.Te freeze-dried material was extracted with boiling acetone, and the residue was concentrated at atmospheric pressure.Tis concentrated residue was extracted successively with light petroleum ether (40-60 °C) and benzene to remove nonfavonoid and other matter.Te dry material, made of favonoids (C6-C3-C6 compounds), was dissolved into a solvent solution with 1% Tween 80.
2.1.3.Determination of Total Flavonoid Content.Te total favonoid content of the extract of leaves from T. diversifolia was quantifed by following the procedure described by Krishnadhas et al. [5].Into a test tube, 1 mL of the plant extract was put, and to it, 0.6 mL of sodium nitrite (5% w/v), 0.5 mL of aluminium chloride (10% w/v), and 3 mL of sodium hydroxide (4.3% w/v) were added and the mixture was added up with distilled water to make 10 mL.Te reaction mixture was left to stand for 15 minutes, after which its absorbance was read at 500 nm in a semiautomated spectrophotometer.Quercetin was used as the standard, and the results were calculated as quercetin equivalence (quercetin eq., mg/ml) of the plant.

Collection of Blood Samples for In Vitro Sensitivity Test.
A human blood sample was collected through venipuncture into an ethylenediaminetetraacetic acid (EDTA) tube and after washing three times by adding normal saline to the blood sample.After a centrifugation at 5000 RPM for 10 min, the supernatant was discarded and cells were washed three times with Roswell Park Memorial Institute Medium-1640 (RPMI-1640).Cells were suspended in RPMI-1640 to make a 20% erythrocyte suspension before the culture.RPMI-1640 was supplemented with l-glutamine (4.2 mM), piperazineethanesulfonic acid (HEPES) (25 mM), bovine foetal serum (10% (v/v)), and streptomycin (100 IU/mL) (complete RPMI-1640).

Harvesting of Murine Peritoneal Cells.
Murine peritoneal cells were harvested from 4 male rats of 6 weeks old.Twenty-four hours before harvesting, rats were intraperitoneally injected with a volume of 1 ml of 25% starch solution to increase the number of cells in the peritoneal cavity.Rats were euthanised by cervical dislocation and the outer skin was cut to expose the inner skin in the peritoneal cavity.A volume of 5 ml of cold PBS containing 2.5% foetal bovine serum (FBS) was carefully injected into the peritoneal cavity (without puncturing any organs).Using a 10 ml syringe, cells were withdrawn and put into tubes and kept on ice.Te collected cell suspension was washed by centrifugation (1800 tr/min, 10 min).Te supernatant was discarded, and the cells were resuspended in RPMI.Te viability of the cells (98.7%) was determined by mixing a volume of 100 μl with an equal volume of 0.4% trypan blue staining dye and counted manually using a Malassez haemocytometer counting chamber under a light microscope.To investigate on this issue, the phagocytic activities of T. diversifolia favonoids were assessed in albino rats (Wistar strain) weighing 200-250 g and of age range 8-10 weeks obtained from the laboratory of physiology and pharmacology of the University of Dschang.Tey were divided into groups of fve.Group I was made of normal rats (normal control) and no treatment was given.Group II was made of rats receiving a dose of methylprednisolone sodium succinate (MPSS) 40 mg/20 ml (1 ml/kg) and received distilled water (negative control).Group III contained MPSSinduced immunocompromised rats and received levamisole (2.5 mg/kg) and was taken as positive control.Groups IV and V (test groups) were made of MPSS-induced immunocompromised rats and were treated with 50 mg/kg and 100 mg/kg of the favonoid extract, respectively.Extract and levamisole were administered via intragastric route by gavage for one week every 2 days.All animals have received on the frst day of treatment an injection of Bacillus Calmette-Guérin (BCG).All animals were kept at room temperature (12 hours cycle of light and darkness) and fed standard feed and had free access to water (ad libitum).

Cytotoxicity Test Using Haemolysis Assay.
Te haemolytic efect of the favonoid extracts of T diversifolia was evaluated in human erythrocytes as reported by Lópezet al. [7].In brief, 5 mL of normal saline was dispensed into 3 test tubes.Tereafter, 5 mL of the extract was added to the frst test tube and a 2-fold dilution was done.A tube containing 5 ml of 1% Tween 80 was taken as a baseline haemolytic control.Ten, 5 mL of the washed RBCs (20% erythrocyte suspension) was dispensed into all the test tubes.Te tubes were incubated at 37 °C for 1 hr and the amount of haemoglobin produced was determined by reading the optical density using a spectrophotometer at 540 nm.Te following formula was used to calculate the percentage of haemolysis: % Haemolysis � OD sample OD control   × 100. (1)

Cytotoxicity Test on Murine Cells Using MTT Assay.
Murine cells (1.5 × 106 cells/ml) were plated onto 96-well plates and incubated with the favonoid extracts (6.3, 12.5, 25, and 50 μg/ml) for 48 h at 37 °C in incubator using the candle as sources of CO2.Cells pated in RPMI complete media only were taken as control, and cells cultured in presence of Tween 80 (15%) were used as positive control.
After the 48 h incubation, 25 μl of MTT solution was added to each well.Te plate was incubated for 3 hours and 50 μl of dimethyl sulfoxide was added to each well.Te optical density (absorbance) was measured at 592 nm using a microplate.

In Vitro Cultivation of P. falciparum Isolates and Sensitivity Test.
To study the sensitivity of P. falciparum against plant favonoid extracts, an infected blood sample was freshly obtained from a P. falciparum-infected donor and was diluted with fresh uninfected human RBCs to have a parasitemia of 8000 P. falciparum/µl of blood.P. falciparum was cultured in 96-well plates as described by Das et al. [8].Te parasites were cultured in the presence of the favonoids of T. diversifolia at diferent concentrations (0.05, 0.025, 0.0125, 0.0063, 0.0031, 0.0016, 0.0008, and 0.0004 mg/mL) in a fnal volume of 200 mL.In detail, plant favonoid extract was dissolved in complete RPMI-1640 supplemented with 0.5% (v/v) DMSO and 0.1% (v/v) Tween 80. Trough a two-fold serial dilution carried out across the plate, the extract at diferent concentrations was distributed in triplicate in 100 µl.A reference drug, Combiate 80/480 mg/mL (artemether and lumefantrine 80/480 mg/ml tablet) dissolved in RPMI-1640, was used as positive control.
Later, the suspension of parasitized RBCs (100 µL) was added into the wells of the microplate.Te plate was then incubated at 37 °C in an incubator for 48 hrs.Te parasitemia was determined using Giemsa stained thick smear on slides after 24 hrs and 48 hrs.To assess the efect of the extract, 20 µL of the culture was collected from each well and used to prepare a thick smear.Te flms were stained with 10% Giemsa solution (pH, 7.3) and observed under the light microscope (100x).Te number of trophozoites was counted in 5 felds.Te percentage of inhibition or inhibitory efect was calculated as follows: where S � (No. of trophozoites in the test well/No. of trophozoites in control well) × 100.

Four-Day Suppressive Test against P. berghei in Mice.
Te test was carried out following the method reported previously [9].Te animals received intraperitoneally a standard inoculum of 1 × 10 7 P. berghei-infected erythrocytes on day zero (D0) and then divided into groups of fve Scientifca animals of 6 animals each.Te doses of the extract were given to them orally for four consecutive days by gavage with the aid of feeding cannula.One group of mice was given 5 mg/kg/day chloroquine (positive control) and another group taken as control group received an equivalent volume of distilled water.Te three remaining received the extract at diferent concentrations each.On the ffth day of the experiment, thin blood flms were made from each mouse and the parasitemia level was estimated.
2.11.Curative Test against P. berghei in Mice.Rane's test [10] was used to evaluate the curative activity or schizontocidal activity of favonoid-rich extract of T. diversifolia leaves against P. berghei in mice.Mice were inoculated, and after 72 hrs, they were divided into the diferent groups and treated with diferent doses of the extracts, chloroquine (5 mg/kg/day), and distilled water.Animals were treated for 5 days.Ten, the parasitaemia level was monitored during the 5 days posttreatment, while the number of survival animal or the time of death in each group was monitored for 15 days of observation.
2.12.Parasitemia and Survival Percentage.In the in vivo test, the parasitemia was determined by counting the number of infected RBCs (a minimum of fve felds per slide) using a light microscope.Percentage of parasitemia and percent of chemosuppression were calculated using the modifed Peters and Robinson formula [11]: Lastly, the animals were followed for 15 days and their mean survival time (MST) was determined using the following formula [4]:

MST �
Total number of days mice survived Total number of mice . (4)

Plasma Level Determination of Interferon and Tumor
Necrosis Factor in P. berghei-Infected Mice.To explore whether the reduction of the parasitemia is associated to the modulation of immune response by T. diversifolia favonoids, plasma levels of INF-c and TNF-α were measured in blood collected as indicators of proinfammatory response activation at day 4 after infection and treatment.Trough heart puncture, blood was collected from all experimental mice using heparinised tubes and their plasma was collected after centrifugation (3000 rpm, 10 min).Sandwich ELISA commercial kit (Invitrogen ® , Termo Fisher Scientifc, USA) was used to quantify the plasma levels of INF-c and TNF-α.Te assay was carried out following the manufacturer's instructions.Te absorbance was measured by optical density at 450 nm in a Microplate Absorbance Reader.Using the standard curve, INF-c and TNF-α levels were expressed as pg/mL.

Measurement of Phagocytic Index in MPSS-
Immunocompromised Rats.Phagocytic index was assessed using the carbon clearance test as reported previously [12].In this test, each rat was intravenously injected with diluted India ink at 100 µL/10 g body weight.After 2 min (T 1 ) and 10 mns (T 1 ), respectively, blood specimens were collected from the rat retinal venous plexuses and then mixed with 2 mL of 0.1% sodium carbonate.Te absorbance was measured at 600 nm on a double beam spectrophotometer with 0.1% sodium carbonate as the blank.Te liver and the spleen were weighed on an electronic balance and its phagocytic index was calculated as follows: where OD1 was for T 1 and OD2 for T 2 .Phagocytic index � ∛k × A/(B + C), where A is the body weight, B is the liver weight, and C is the spleen weight.

Assessment of Energy Metabolism and Oxygen Metabolite Production in MPSS-Immunocompromised Rats.
After a week of treatment and 24 hrs after the last administration, rats were weighed and then sacrifced using diazepam and ketamine (ratio: 3 : 1).Peritoneal cells were harvested by washing the peritoneum in 3 mL of PBS and used for metabolism assay.Blood was collected by cardiac puncture and the serum was harvested to measure the production of nitric oxide and oxygen radicals.Spleen and thymus were collected and weighed using an electronic balance.Te organ index was calculated using the following formula reported by Evans Ngwenah et al. [13].
Relative weight mg g   � (weight of thymus or spleen) body weight . ( To evaluate the mitochondrial metabolism, a cell suspension (density: 1 × 10 3 peritoneal cells/ml) was prepared and 2 ml of this suspension was mixed with 1 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (1% in PBS).After 2 hrs of incubation, 1N HCl (1 ml) was added and the optical density was read at 540 nm.Te mean of the optical densities in treated animal 4 Scientifca was compared to that of control to evaluate the energy metabolism of the cells [13].In addition, nitrite oxide production was measured using the Greiss reagent as reported by Mahamatet al. [12].Serum sample (100 µL) was mixed in equal volumes with the Griess reagent (mixture of sulfanilamide solution and N-1-naphthylethylenediamine dihydrochloride solution).Te absorbance at 540 nm was then measured using a microplate spectrophotometer (VersaMax), and the concentrations of the nitrite were obtained using the standard curve obtained using NaNO 2 .
Te protocol reported by Pinakiet al. [14] was used to assess the level of reactive oxygen species.In brief, 100 µl of plasma diluted 20 times in phosphate bufer was mixed with 1 mL of acetate bufer.Tereafter, 25 µL of working chromogen solution (N, N-dimethyl-p-phenylenediamine sulphate, Aldrich, Sigma) was added and the absorbance was taken at 505 nm after 6 min using a spectrophotometer.Te absorbance values obtained were compared to the curve obtained using H 2 O 2 .

Statistical Analysis.
Experimental values were expressed as mean ± standard deviation of an experiment done in triplicate.Data were analysed using one-way analysis of variance, followed by post hoc Student's t tests.Levels of p < 0.05 were considered as indicative of signifcance.All calculations were carried out using the GraphPad Prism ® V5.03 software (GraphPad Software Inc ® , CA, USA).

Cytotoxicity Efect of T. diversifolia Flavonoids-Rich Extract against
Red Blood Cells.Te preliminary screening carried out on the cytotoxic efect of the favonoid-rich extract of T. diversifolia leaves against red blood cells revealed a signifcant cytotoxic efect (Table 1).All the tested concentrations of the extract caused a red blood cell haemolysis in a concentration-dependent manner (p < 0.05).A percentage of 28.83% was observed with the highest tested concentration of 0.05 mg/mL.

In Vitro Cytotoxicity Assay Using Rat Peritoneal Cells.
Te favonoids-enriched extract obtained from T. diversifolia leaves was tested for cytotoxic activity against immune cells obtained from rat's peritoneal cavity.Te results of the MTT assay plate as presented in Figure 1 showed that T. diversifolia had no signifcant cytotoxic activity (p > 0.05) when compared to results for cells in medium only.

In Vitro Study of Susceptibility of P. falciparum to T. diversifolia Flavonoids-Rich Extract.
Te results of the favonoid-rich extract of the T. diversifolia leaves on the P. falciparum during the in vitro test suggested that all the tested concentrations of the extract had a signifcant (p < 0.0001) inhibitory efect against the trophozoite stage after 24 hrs and 48 hrs (Table 2).Te favonoid-rich extract of T. diversifolia signifcantly decreased the parasite load in a concentration-dependent manner.Tested concentrations from 0.0031 mg/mL showed similar efect like the reference drug (artemether 80 mg/lumefantrine 480 mg) at 5.6 mg/ml (p > 0.05).

Suppressive Efect of T. diversifolia Flavonoids-Rich
Extract.As shown in Table 3, the favonoid-rich extract of T. diversifolia leaves showed a signifcant efect (p < 0.001) during the 4-day suppressive test.In comparison with the negative control group, the plant favonoid extract signifcantly (p < 0.05) reduced the parasitemia in a dose-dependent manner.But still the standard drug (5 mg/kg/day) is more efective than the highest doses (100 mg/kg/day), which showed a chemosuppression of 66.40% fractions (p < 0.05).

Curative Efect of T. diversifolia Flavonoids-Rich Extract.
Accordingly, the test doses of the favonoid-rich extract of T. diversifolia leaves showed a signifcant efect (p < 0.001).
In comparison with the negative control group, all the test doses of the extract reduce parasitemia signifcantly (p < 0.05).Besides, a dose-dependent parasitemia suppression efect was also observed.Similarly, the three extract doses prolonged the survival time of parasite-infected mice signifcantly (p < 0.001) in comparison with the negative control groups.However, the efect of the extract was lower than that of the group treated with artemether 80 mg/ lumefantrine 480 mg (Table 4).

Efect of T. diversifolia Flavonoids-Rich Extract on Plasma Levels of INF-c and TNF-α in Mice Infected with P. berghei.
Te result indicated that the groups of animals who were given the Tithonia diversifolia favonoids (50 and 100 mg/kg) and artesunate had a signifcantly high plasma level of INF-c compared to infected animals not treated (Figure 2(a)).
Similarly, in all groups of animals taking the Tithonia diversifolia favonoids and artesunate, the TNF-α plasma levels were signifcantly higher than those in animals infected but not treated (Figure 2(b)).

Phagocytic Efect of Flavonoids-Rich Extract of T. diversifolia in MPSS-Immunocomprised Rats.
Te efect of the administration of favonoids in MPSS-treated rats is presented in Table 5. Treatment with the extract signifcantly increased the size of the spleen as compared to the negative control.At 100 mg/kg, the efect of the favonoid extract was similar to that of BCG vaccine.In contrast, the treatment with the favonoid extract as well as the BCG did not afect the thymus index.Furthermore, treatment of the rats with favonoid-rich extract increased the phagocytic index of the test animals compared to the animals that were treated with immunosuppressive drugs.Te phagocytic index at 100 mg/kg was similar to that of animals treated with BCG vaccine.Treatment of rats with favonoid-rich extract increased the cell metabolism of the animals compared to those kept without any treatment.At 50 mg/kg, the efect of the favonoid extract was higher than that of BCG vaccine, and the results were signifcant at p ≤ 0.004.Also, favonoid-   6 Scientifca rich extract of leaves of T. diversifolia signifcantly increased the serum NO of rats in a dose-dependent manner within one week of the treatment in rats compared to the serum level in control group.Similarly, injection of BCG increased serum level of NO in the rats.Te total oxygen radical level in the serum of rats treated with the favonoid-rich extract from T. diversifolia increased in a concentration-dependent manner.BCG was injected to the animals, and the level of oxygen radicals was increased compared to the serum level of oxygen radicals of the negative control (Table 5).

Discussion
Bioactive compounds found in the crude extract of the plant could produce their antimalarial efect via diferent mechanisms.Flavonoid compounds have been found to inhibit the growth and multiplication of the parasite that are essential for the control of the infection [15,16].Besides, phytochemicals like favonoids and others may also exert their antimalarial efects indirectly by stimulating the immune system of the host [17].Currently, there are no recommendations regarding the optimal content of favonoids of T diversifolia and their subclasses in malaria treatment.However, many studies have evaluated the antimalarial activity of plant derivatives and indicated their efcacy against the Plasmodium species using in vivo and in vitro methods [18][19][20].In the present study, the antimalarial efect of the favonoid-rich extract of Tithonia diversifolia was evaluated by assessing its antiplasmodial and immunomodulatory activities.In the preliminary assay, the plant showed a toxic efect to the red blood cells as demonstrated by the hemolytic efect (28.83%) of the extract at 0.05 mg/ml.However, the extract did not show any toxic efect against murine peritoneal cells.Tis controversial efect may justify the reason why a low dose was recommended by the traditional healers.Despite the fact that the toxicity study reveals its safety, it is most important to perform a well-designed preclinical study to fnd a lead compound with desired efcacy for clinical study.
Te potential antimalarial activities of the favonoid-rich extract of T. diversifolia were evaluated in vitro against P. falciparum and in vivo using mice by evaluating the suppressive tests, standard models for the antimalarial screening [21].Consequently, in the in vitro test, all the test doses of the favonoid-rich extract of T. diversifolia inhibited the growth of trophozoite at 24 and 48 hrs in a dose-dependent manner, with the percentage of inhibition varying from 19 to 100%.Terefore, the plant favonoid could have the potential of antiplasmodial activity [22,23], specifcally against the erythrocytic stages of the parasite.Besides, all the test doses of the favonoid-rich extract of T. diversifolia inhibited the level of parasitemia and improved the survival time of infected mice in a dose-dependent manner in 4-day suppressive test as well as in Rane's test.Terefore, the favonoid extract of T. diversifolia could have schizontocidal activity and could be used in preventive and curative treatment of malaria infection.Te results of this study are in agreement with those of Afolayan et al. [24] who demonstrated the antimalarial efect of T. diversifolia in combination with other plants.Te results showed that the percentage suppression was relatively lower during established infection (Rane test) than its efect in the 4-day suppressive test, and the favonoid extract of leaves of T. diversifolia still showed a promising curative potential in established infection, justifying the probable rapid action of the extract during Rane's test.Taken together, the results of Rane's test suggest that the favonoid-rich extract of leaves of T. diversifolia has therapeutic potential against established malaria infection since it is desirable to have both suppressive and curative activities in a phytodrug [21].In both models (4-day suppressive test and Rane's test), the percentage suppression of parasitemia is ≥30% and such compounds are considered active [25].

Scientifca
In this study, TNF and INF levels, measured on day 4 after infection, showed high levels in infected mice receiving the extract.Tis may indicate that T. diversifolia favonoid-rich extract stimulates specifc patterns of innate immune response to infection with P. berghei.In various mouse strains, studies carry out with P. yoelii showed that IFN-c and TNF produced 24 h after infection by cδ T lymphocytes and natural killer cells are indispensable for macrophage activation and early control of parasitaemia [26].Tis may justify the reduction of the parasitemia in mice.
Studies demonstrated that when infected blood was inoculated in healthy mice, elimination of Plasmodiuminfected erythrocytes occurs mainly in the spleen by monocyte-macrophage [27].In this study, the administration of T. diversifolia favonoid-rich extract along with the injection of BCG resulted in enhanced weight of spleen in immunocompromised rats.Tis demonstrates that the favonoid extract of T. diversifolia can be helpful to boost the immune response.Te plant favonoid could have the potential of immunostimulatory activity.Spleen and thymus indices are considered to be the most elementary and conventional indices, which have been generally greatly exploited to evaluate the whole immune state of the organism [28], and compounds can be considered immunostimulatory active when there is increase in the indices of such organs [29,30].Consequently, the favonoid-rich extract of leaves of T. diversifolia was presumed to be active.Besides, it was observed that peritoneal cells (neutrophils, monocytes, and macrophages) of mice treated with the favonoid-rich extract of T. diversifolia have high metabolic and phagocytic activities.As phagocytes are the main participants in the innate immune response among the earliest cell types to respond to invasion by pathogenic organisms [31,32], it may be intriguing that favonoid-rich extract of leaves of T. diversifolia can be helpful to stimulate the immune response and consequently control the infectious diseases such as malaria infection [12].Additionally, the results of the study showed that the serum has high concentration of NO and oxygen radicals in methylprednisolone-immunocompromised rats treated with the extract.NO and oxygen radicals are part of substances produced during intracellular killing mechanism in phagocytes.Terefore, these results are additional evidence demonstrating that the favonoid-rich extract of T. diversifolia could have immunostimulatory activity and could be used in preventive and curative treatment of malaria infection.

Conclusion
According to this study, it can be concluded that that the favonoid-rich extract of T. diversifolia is a good candidate to use in treatment of malaria as demonstrated here by its schizontocidal, antimalarial, and immunostimulatory activities.In this study, the crude favonoids-rich extract was used.Tis may not provide the real efcacy of the extract.More investigations must be performed to assess the safety and use of T. diversifolia favonoids and to identify the concerned types of favonoids.

Figure 1 :
Figure 1: Efects of favonoid-rich extract of leave from T. diversifolia on the viability of mouse peritoneal cells using the MTT assay.Results are expressed as mean ± SD and Turkey's multiple range test (p < 0.05) was used to compare the groups.* * * p < 0.001.

Table 1 :
Haemolytic percentage of favonoid-rich extract of T. diversifolia leaves against human red blood cells.

Table 2 :
Inhibition percentage of favonoid-rich extract of T. diversifolia leaves against P. falciparum at 24 hrs and 48 hrs of incubation.Data are expressed as mean ± SD (test done in quadruplicate).Superscript letters indicate the diference within the group following Student's post hoc test with p < 0.05.Positive control: artemether 80 mg/lumefantrine 480 mg (5.6 mg/ml).

Table 3 :
Parasitemia and survival time of infected mice treated with the favonoid-rich extract of T. diversifolia leaves in the 4-day suppressive test.Data are expressed as mean ± SD (n � 6).Superscript letters indicate the diference within the group following Student's post hoc test with p < 0.05.Positive control: infected mice receiving artemether 80 mg/lumefantrine 480 mg (5 mg/kg); Neg.control: infected mice receiving distilled water.

Table 4 :
Parasitemia and survival time of infected mice treated with favonoid-rich extract of T. diversifolia leaves in the 4-day suppressive test.Data are expressed as mean ± SD (n � 6).Superscript letters indicate the diference within the group following Student's post hoc test with p < 0.05.Positive control: infected mice receiving artemether 80 mg/lumefantrine 480 mg (5 mg/kg); Neg.control: infected mice receiving distilled water.

Table 5 :
Spleen and Tymus Index, Phagocytic Index and Serum Level of Total Oxygen Radicals and Nitric Oxide (NO) in Rats Treated with the Flavonoid-Rich Extract of T. diversifolia leave.
control: non-immunosuppressed animals without any treatment ; neg.control: immunosuppressed animals treated with distilled water only.